|Year : 2022 | Volume
| Issue : 2 | Page : 126-130
Determining the probability of malignant transformation of tobacco-induced oral leukoplakia using tissue p53 as a prognostic marker – A cross-sectional study
Bhargavi Ramesh1, Nalini Aswath1, Vidhya Rani Shyamsundar2
1 Department of Oral Medicine and Radiology, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Department of Oral and Maxillo Facial Pathology, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India
|Date of Submission||21-Jan-2022|
|Date of Decision||31-Mar-2022|
|Date of Acceptance||20-May-2022|
|Date of Web Publication||22-Jun-2022|
Department of Oral Medicine and Radiology, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Chennai - 100, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: The concept of field cancerization necessitates substantiation of clinical assessment and histopathological examination by molecular markers. Molecular tumor biomarkers such as p53 protein overexpression aid in assessing the malignant transformation of such potentially malignant oral lesions. Aim: To identify the molecular changes in oral leukoplakia by studying the immunoexpression of tissue p53 and correlating it with the clinical and histological findings to establish an appropriate treatment plan. Methods and Material: Biopsy samples taken from 25 oral leukoplakia cases were subjected to histopathological and immunohistochemical analysis to determine the percentage positivity of p53 expression. Histopathological grading was based on dysplastic features and level of epithelial involvement. Dysplasia grading in IHC sections was estimated by p53% positivity in the cells. The obtained data were subjected to statistical analysis. Results: 24 samples were considered for statistical analysis due to the loss of epithelium in the IHC section of one sample. The results showed a statistically significant association between histopathological and IHC grading of dysplasia with a P value less than 0.05 in Fisher Exact test. Conclusion: Increased expressions of p53 in potentially malignant oral lesions are proportional to the risk of malignancy. The gold standard histopathological result does not reveal the molecular abnormality associated with a potentially malignant oral lesion. Therefore molecular analysis of such lesions will aid in the effective prevention of oral cancer.
Keywords: Immunohistochemistry, oral leukoplakia, p53, potentially malignant
|How to cite this article:|
Ramesh B, Aswath N, Shyamsundar VR. Determining the probability of malignant transformation of tobacco-induced oral leukoplakia using tissue p53 as a prognostic marker – A cross-sectional study. J Indian Acad Oral Med Radiol 2022;34:126-30
|How to cite this URL:|
Ramesh B, Aswath N, Shyamsundar VR. Determining the probability of malignant transformation of tobacco-induced oral leukoplakia using tissue p53 as a prognostic marker – A cross-sectional study. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Sep 25];34:126-30. Available from: https://www.jiaomr.in/text.asp?2022/34/2/126/347924
| Introduction|| |
Oral cancer ranks one of the top three cancers in India. Oral squamous cell carcinomas [OSCC] are the most common types of oral cancers. Widespread usage and easy accessibility to tobacco products in our country are major contributors to the cancer burden. Carcinogenesis isn't always a de-novo process. Premalignant disease in patients with increased risk factors indicates future cancer progression. Early detection and prompt treatment have always proven to be more beneficial in managing oral cancers. While multiple oral precancerous lesions have been described in the literature, oral leukoplakia is the most commonly encountered lesion. As per standard definition, oral leukoplakia is defined as “predominantly white plaques of questionable risk, having excluded (other) known diseases or disorders that carry no increased risk for cancer.” Biopsy remains the gold standard investigation in identifying and assessing the severity of dysplasia of oral leukoplakia. While dysplasia depicts the altered cellular architecture of the affected tissues, identifying underlying molecular changes aids in understanding the fundamental nature of the disease. Cancer biology and molecular oncology have diversified tumor therapeutics and predict a tumor's clinical behavior. The need for tumor biomarker evaluation and other regular investigations is advantageous to both patients and the advancement of science. Various scientific researchers have established a strong correlation between the altered tumor suppressor gene and tumorigenesis in the human body. The p53 gene is a nuclear transcription factor located on chromosome 17 along its short P arm. This gene regulates the cell cycle by programming senescence and induction of apoptosis. The p53 oncogene plays a major role in DNA replication and repair, thus maintaining genomic stability. This multifunctional aspect of the p53 gene increases the risk of mutation. Thus, the altered expression of p53 is associated with more than 50% of human body cancers. The tissue immunoexpression of the P53 gene is identified by the accumulation of altered protein inside the nucleus of the affected cells. An immunohistochemical (IHC) evaluation of potentially malignant tissue for the presence or absence of p53 expression can detect malignant transformation and patient-specific tumor risk assessment. Overexpression of p53 is associated with the chemo-resistant nature of the tumor. Therefore, a study was hypothesized based on the assumption that immunohistochemical analysis of oral leukoplakia will show increased p53 expression proportional to the dysplastic changes noted in histopathology.
| Materials and Methods|| |
A cross-sectional study involved 25 patients aged above 18 years (22 males and 3 females) with tobacco-associated oral leukoplakia. Institutional ethics committee approval [SBDCH/IEC/04/2019/15] was obtained. Informed consent was obtained from all the participants for participating in the study. Patients of age above 18 of either gender who reported to our OPD with tobacco habits with a presence of oral leukoplakia lesions were included in the study. Patients with bleeding disorders who were contraindicated for biopsy were excluded. Tobacco cessation counseling was given to all the participants. The patients were further advised to apply topical antifungal agents thrice daily for one week. The patients were reviewed, and an incisional biopsy of the oral leukoplakia lesion was done. The biopsy tissue was subjected to histopathological and immunohistochemical analysis. The primary antibody used in the study to detect tissue p53 was a mouse monoclonal antibody. The PolyExcel HRP/DAB detection system was the secondary antibody – a Two-step Universal kit (PathnSitu) for mouse and rabbit primary antibodies. All the samples were analyzed for the following parameters:
- Dysplasia grading in HP
- Epithelial thickness in (μm) [Figure 1]
- P53% (Number of positive cells per 100 cells)
- Level of epithelial involvement (Basal, parabasal, spinosum, and keratin) [Figure 2]
- Intensity of stain uptake on IHC (1- mild uptake, 2- moderate uptake, 3- intense uptake)
- P53 grading [Based on % p53: 0 = 0 (%), 1 = 1-10 (%), 2 = 11-25 (%), 3 = 26-50 (%), 4 = more then 50(%)]
- IHC score (p53%grade x intensity)
- Final score [FS] (1 = 1-3 of IHC score, 2 = 4-6 of IHC score, 3 = 7-9 of IHC score, 4 = 10-12 of IHC score)
|Figure 1: An immunohistochemistry slide of oral epithelium showing the measurement of epithelial thickness in (μm)|
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|Figure 2: A pie chart showing the level of epithelial involvement (Basal, parabasal, spinosum, and keratin)|
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A Microsoft Excel spreadsheet was used for data collection to identify the relationship between tissue p53 expression and the severity of dysplasia in the IHC samples. Statistical calculation was performed on IBM SPSS software (Version 23), and the results were obtained. Those cases which showed no dysplasia in HPE were advised to apply topical Vitamin A on the lesional site (Cap. Aquasol A, twice daily for one month) and systemic antioxidant (Tab. Antoxid OD for three months). Those cases which showed dysplasia in HPE were advised to complete laser excision. They were followed up for the next six months and reinforced for complete habit cessation.
| Results|| |
The study sample consisted of 25 histopathological samples, out of which only 24 could be assessed for IHC, as one sample had a loss of epithelium on IHC sections. The site of distribution of oral lesions is depicted in [Table 1]. In this study, we have categorized the lesions with no dysplasia to mild dysplasia as low-risk category and lesions with moderate dysplasia to severe as high risk or prominent dysplastic lesions [Table 2]. Out of 24 cases analyzed, 7 cases showed a discrepancy between the histopathological and IHC reporting of the grade of dysplasia as seen in [Table 3]. i.e., 1 case of no dysplasia in HPE was reported as mild dysplasia in IHC. 11 cases of moderate dysplasia in HPE were reported as mild -3, moderate-6, severe-1, and oscc-1 in IHC, and 1 case of severe dysplasia in HPE was reported as OSCC in IHC. [Table 3]. This discrepancy was subjected to statistical analysis. Fisher's test was performed between the test parameters. [Table 4] Fisher's exact test P value was less than.05
|Table 1: Shows the Average Distribution of the Lesion Among the Various Sites|
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|Table 3: Shows The Discrepancy in HP Reporting of Degree of Dysplasia Vs IHC Final Score|
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A significant association was observed between histopathological reporting of dysplasia (based on the structural and morphological abnormalities) and IHC grading (based on a percentage of tissue p53 expression). P53 grading for risk assessment of malignancy is independent of histopathological grading.
| Discussion|| |
Determining the malignant transformation of a precancerous oral lesion is tedious. Predicting the transformation of a single clinical lesion requires considering multiple deterministic factors and a sincere follow-up. One of the major factors that determine the malignant potential of these lesions in our regular clinical practice is oral epithelial dysplasia (OED). Histopathological reporting of OED is a subjective analysis dependent on the reporting pathologist. Moreover, not necessarily all dysplastic lesions turn malignant; some may undergo spontaneous regression, or worse, a non-dysplastic lesion can become malignant.,
Therefore, identifying other suitable predictive markers and correlating them to the current gold standard criteria is essential. Evaluation of high-risk lesions with a two-tier histopathology system with immunohistochemistry will produce stable and reliable results that will improve surveillance planning and further management. Literature evidence shows a proportional increase in malignant transformation rate as the lesions progress from mild to moderate dysplasia, 10.3%, and severe dysplasia to carcinoma-in-situ is 24.1%. Interestingly, out of 24 cases in this study, 2 cases reported as moderate and severe dysplasia in histopathological reporting turned out to be early invasive squamous cell carcinoma with basement membrane breach in the IHC sections. This is a perfect example of subjective variation in the reporting of dysplasia. The nuclear staining technique used in IHC detection of p53 expression enabled a better visualization of cells.
Clinical risk assessment factors
Oral leukoplakia has a peak incidence in the 4th to 5th decade of life. However, our study showed an equal number of cases in the 3rd, 4th, and 5th decades. This might be due to the smaller sample size of the study. The malignant potential of oral leukoplakia increases with age as the duration of the lesion present in the mouth, and prolonged habit association also increases. Therefore, older age group people are more susceptible to malignant transformation of leukoplakia when compared to younger age patients. The distribution of Oral leukoplakia among the sexes is higher in men than in women. The men: women ratio is reported to be 2:1. The risk of developing cancer is known to be higher in women when compared to men. The association between tobacco usage and oral cancer is a known fact. The majority of the literature supports a high risk of malignancy in tobacco-associated lesions. However, few studies indicate that non-tobacco associated lesions that are idiopathic are more prone to malignant transformation. These lesions might occur due to chronic irritation or HPV association. Narayan et al. conducted a meta-analysis of clinicopathological risk factors to predict the malignant transformation rate in oral leukoplakia. He reported an equal risk of cancer in both smokers and non-smokers. Tobacco association in any form remains evident for the increase in the risk of cancer. All the patients included in this study were tobacco users in either smoking or chewing. The literature evidence shows a direct association of the role of tobacco in the mutation of the p53 gene, which makes the lesions more prone to carcinogenesis. Dionne et al. conducted a systematic review which says younger populations without habit etiology are more prone to malignant transformation than the older age group. Based on the anatomical location of the lesion, the risk for malignant transformation varies. While buccal mucosa remains the most common site of occurrence, the risk for malignancy is higher in lesions on the floor of the mouth, followed by tongue lesions. The smoking-associated floor of the mouth lesions shows an increased risk as the epithelial thickness is least in that region. However, in the cases of non-smokers, idiopathic tongue lesions carry a greater risk of cancer. The present study is by the literature, with the incidence of buccal mucosa lesions being the highest (68%), followed by the retromolar region (12%) and the tongue (8%), as shown in [Table 1]. The significance of the epithelial thickness is based on the anatomical site of the lesion. The normal oral mucosal epithelium is thickest in the buccal mucosa region (659.79 μm) and thinnest at the floor of the mouth region (100.07 μm) and thus, making it more susceptible to cancer invasion.
In our study, a correlation between the epithelial thickness and the lesion site was done [Figure 3]. The greatest epithelial thickness was found in the lower retromolar pad area, which was reported in HP as moderate dysplasia. However, a biopsy sample from that region turned out to be malignant in the IHC section with the reported epithelial thickness of 873 μm. Another such lesion in the current study, sampled from the buccal mucosa, was reported as severe dysplasia in HP. But on IHC analysis, the epithelial thickness was 825 μm, and it was graded as early invasive squamous cell carcinoma [Figure 4]. An abnormal increase in epithelial thickness suggests an invading tumor front with a finger-like projection of rete ridges into the underlying connective tissue. Assessment of this epithelial depth gives an insight into a missed diagnosis during routine histopathological grading. Visualization of epithelial thickness was easy in IHC sections as the analysis was done using the nuclear staining method, which enables a better picture of the epithelial cells.
|Figure 3: Bar graph showing a correlation between the epithelial thickness and the lesion site|
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|Figure 4: An immunohistochemistry slide of oral epithelium showing early invasive squamous cell carcinoma with epithelial thickness measuring to be 825 μm while the histopathology reported severe dysplasia|
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P53 Expression in oral potentially malignant disorders
Tissue p53 expression is directly proportional to the degree of dysplasia. The overexpression of this protein has confirmed an increase in its malignant potential. Along the malignant transformation process, the role of p53 remains to be in the evolution of dysplasia to early invasive oral cancer. Increased expression of p53 indicates an active disease status irrespective of the structural and architectural changes of the cells. Cruz et al. reported that p53 could be used as a risk marker for dysplasia if not a prognostic marker for malignant transformation. In our study, 50% of the cases (12 out of 24) showed p53 expression till the keratin layers of the epithelium on IHC sections, indicating active disease status [Figure 2].
Our study showed a significant association between histopathological grading of dysplasia and the percentage of p53 expression. We recommend the following steps in evaluating the risk of malignant transformation in oral leukoplakia.
- Histopathological reporting of dysplasia along with the grading of %p53 expression
- Assessment of epithelial thickness for excessive increase.
- Clinicopathological correlation of the lesion based on other determinants.
The following suggestions may also be considered during the review
- Reduction in duration between the visits during the surveillance period.
- Sequential biopsy in the high-risk group to monitor any progression.
- Identification of co-expression markers (Ki67, VEGFR) expression in a lesion and their synergistic effect in tumorigenesis to better understand tumor behavior.
| Conclusion|| |
Evaluation of leukoplakia with a two-tier system of histopathology and immunohistochemistry can help the pathologist diagnose increased dysplastic activity and microinfusion of tumor cells within the connective tissue. P53 overexpression in epithelial cells indicates a higher risk of malignant transformation in oral leukoplakia. This will be helpful in disease surveillance, treatment planning, predicting the outcome, monitoring the efficacy of therapeutic agents intended to reverse potential carcinogenesis in these lesions, and evaluating newer developing topical anticancer drugs.
Since a positive association has been reported in such a small sample size, it is desirable to conduct the study in a larger population with a bigger sample size in the future to establish p53 expression as a definite prognostic marker for malignant transformation.
The authors wish to thank the department of molecular Oncology, Cancer Institute, Adyar, for providing the infrastructure needed for the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45:309-16.
Coelho KR. Challenges of the oral cancer burden in India. J Cancer Epidemiol 2012;2012:701932.
Chaturvedi AK, Udaltsova N, Engels EA, Katzel JA, Yanik EL, Katki HA, et al
. Oral leukoplakia and risk of progression to oral cancer: A population-based cohort study. J Natl Cancer Inst 2020;112:1047-54.
Warnakulasuriya S. Clinical features and presentation of oral potentially malignant disorders. Oral Surg Oral Med Oral Pathol Oral Radiol 2018;125:582-90.
Rajguru JP, Mouneshkumar CD, Radhakrishnan IC, Negi BS, Maya D, Hajibabaei S, et al
. Tumor markers in oral cancer: A review. J Family Med Prim Care 2020;9:492–6. [Full text]
Mathur P, Sathishkumar K, Chaturvedi M, Das P, Sudarshan KL, Santhappan S, et al
. Cancer statistics, 2020: Report from national cancer registry program, India. JCO Global Oncol 2020;6:1063-75.
Ozaki T, Nakagawara A. Role of p53 in cell death and human cancers. Cancers 2011;3:994-1013.
Gougis P, Moreau Bachelard C, Kamal M, Gan HK, Borcoman E, Torossian N, et al
. Clinical development of molecular targeted therapy in head and neck squamous cell carcinoma. JNCI Cancer Spectrum 2019;3:pkz055.
Fischer DJ, Epstein JB, Morton TH Jr, Schwartz SM. Interobserver reliability in the histopathologic diagnosis of oral pre-malignant and malignant lesions. J Oral Pathol Med 2004;33:65-70.
Reibel J. Prognosis of oral pre-malignant lesions: Significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med 2003;14:47-62.
Pitiyage G, Tilakaratne WM, Tavassoli M, Warnakulasuriya S. Molecular markers in oral epithelial dysplasia. J Oral Pathol Med 2009;38:737-52.
Ranganathan K, Kavitha L. Oral epithelial dysplasia: Classifications and clinical relevance in risk assessment of oral potentially malignant disorders. J Oral Maxillofac Pathol 2019;23:19-27.
] [Full text]
Mehanna HM, Rattay T, Smith J, McConkey CC. Treatment and follow-up of oral dysplasia—A systematic review and meta-analysis. Head Neck 2009;31:1600-9.
Gopinath D, Thannikunnath BV, Neermunda SF. Prevalence of carcinomatous foci in Oral leukoplakia: A clinicopathologic study of 546 Indian samples. J Clin Diagn Res 2016;10:ZC78-83.
Dionne KR, Warnakulasuriya S, Binti Zain R, Cheong SC. Potentially malignant disorders of the oral cavity: Current practice and future directions in the clinic and laboratory. Int J Cancer 2015;136:503-15.
Narayan TV, Shilpashree S. Meta-analysis on clinicopathologic risk factors of leukoplakias undergoing malignant transformation. J Oral Maxillofac Pathol 2016;20:354-61.
] [Full text]
Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 2002;21:7435-51.
Schepman KP, Bezemer PD, Van Der Meij EH, Smeele LE, Van Der Waal I. Tobacco usage in relation to the anatomical site of oral leukoplakia. Oral Dis 2001;7:25-7.
Cruz IB, Snijders PJ, Meijer CJ, Braakhuis BJ, Snow GB, Walboomers JM, et al
. p53 expression above the basal cell layer in oral mucosa is an early event of malignant transformation and has predictive value for developing oral squamous cell carcinoma. J Pathol 1998;184:360-8.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]